Treatment Of Snakebite Research Articles

#2781 Renal cortical necrosis—a rare cause of acute kidney injury

Abstract Background and Aims Renal cortical necrosis (RCN) is a rare and catastrophic cause of acute kidney injury (AKI). It is characterized by ischemic destruction of all the elements of renal cortex due to marked reduction in renal arterial perfusion of renal cortex due to vascular spasm and microvascular injury. It can be patchy or diffuse depending upon the cause and extent of hypoperfusion. Snake bite is rare cause of RCN. Incidence of RCN following snake bite has been reported to be as high as in 25% of the bitten patients to <1%. Lack or delay in receiving anti snake venom (ASV) has been observed to be a contributing factor in development of RCN following snake bite. Method A 45-year-old female had an unknown bite on foot while returning to home at twilight. She had intense pain at site of bite with slight ooze of blood. Overnight she had vomiting and pain abdomen. Subsequently she developed progressive fall in urine output and dyspnoea. After about 36 hours of bite at the time of admission in hospital she had anuria, prolonged whole blood clotting time, tachypnoea, hypoxemia and required oxygen (O2) to maintain normal O2 saturation. She was given two doses of polyvalent ASV 6 hours apart which corrected her coagulation defect. Investigations showed: haemoglobin—9 gm/dl, total leucocyte count—18700/mcl, platelet—1.15 lakh/mcl, blood urea—88 mg/dl and serum creatinine—10.5 mg/dl. She was managed with haemodialysis and supportive care. Even after 3 weeks of onset of AKI, she had anuria so a kidney biopsy was done. Results kidney biopsy on light microscopy examination showed 9 glomeruli; 8 revealing global tuft necrosis along with necrosis of adjacent tubules and vasculature and necrotic process affected around 60% of sampled cortical area consistent with cortical necrosis (Fig. 1). She was continued with haemodialysis support and urine output and pre-dialysis serum creatinine were monitored. Her urine output after 3 months of onset of AKI is around one litre per day but her pre-dialysis serum creatinine stays in the range of 9-10 mg/dl and so she continues to receive biweekly haemodialysis. Conclusion In our index case, there was delay in getting ASV which may have contributed to development of RCN. So, early treatment of snake bite is important to prevent this catastrophic complication.

Doxycycline-Mediated Inhibition of Snake Venom Phospholipase and Metalloproteinase.

Warfighters are exposed to life-threatening injuries daily and according to the Joint Trauma System Military Clinical Practice Guideline-Global Snake Envenomation Management snakebites are a concerning threat in all theaters of operation. Snake venom is a complex mixture of toxins including phospholipases A2 (PLA2) and snake venom metalloproteinases (SVMP) that produce myotoxic, hemotoxic, and cytotoxic injuries. Antibody-based antivenom is the standard of care but new approaches including small-molecule inhibitors have gained attention in recent years. Doxycycline is an effective inhibitor of human metalloproteinases and PLA2. The enzymatic activities of 3 phylogenetically distinct snakes: Agkistrodon piscivorus, Naja kaouthia, and Daboia russelii were tested under inhibitory conditions using doxycycline. Enzymatic activity of PLA2 and SVMP was measured in N. kaouthia, D. russelii, and A. piscivorus venom alone and with doxycycline using EnzChek Phospholipase A2 and Gelatinase Assay Kits. A 1-way ANOVA with Tukey's post-hoc test was used to conduct comparative analysis. The median lethal dose of the venoms, the effective dose of doxycycline, and creatine kinase (CK) inhibition levels were measured in a murine model with adult Bagg Albino (BALB/c) mice using intramuscular injections. Median lethal and effective doses were determined using Spearman-Karber's method and a 1-way ANOVA with Tukey's post-hoc test was used to compare CK inhibition levels. Phospholipases A2 activity was reduced to 1.5% to 44.0% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline when compared to venom-only controls (P < .0001) (Fig.1A). Snake venom metalloproteinases activity was reduced to 4% to 62% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline (P < .0001) (Fig.1B). The lethal dose (LD50) values of the venoms in the murine model were calculated as follows: A. piscivorus = 20.29 mg/kg (Fig.2A), N. kaouthia = 0.38 mg/kg (Fig.2B), and D. russelii = 7.92 mg/kg (Fig.2C). The effective dose (ED50) of doxycycline in A. piscivorus was calculated to be 20.82 mg/kg and 72.07 mg/kg when treating D. russelii venom. No ED50 could be calculated when treating N. kaouthia venom (Fig.3). Creatine kinase activity was significantly decreased in all 3 venoms treated with doxycycline (P < .0001) (Fig.4). Doxycycline reduced PLA2- and SVMP-related lethality, particularly in A. piscivorus envenomings and in a limited capacity with D. russelii revealing its promise as a treatment for snakebites. In addition, CK activity, a common indicator of muscle damage was inhibited in mice that received doxycycline-treated venom. The doxycycline concentrations identified in the ED50 studies correspond to 1,456 to 5,061 mg dosages for a 70 kg human. Factors including venom yield and snake species would affect the actual dosage needed. Studies into high-dose doxycycline safety and its effectiveness against several snake species is needed to fully translate its use into humans. Based on this work, doxycycline could be used as a treatment en route to higher echelons of care, providing protection from muscle damage and reducing lethality in different snake species.

Perceptions on Indigenous Medicine, First Aid and Treatment of Snakebite among Sri Lankan Farmers

Since snakebite is a serious public health concern in Asia, one of the most affected countries is Sri Lanka. The incidence, species variety, clinical symptoms, and management of snakebite in Asia, with a focus on Sri Lanka, are all covered in this review paper. Many of the venomous snake species found in Asia are abundant and diverse. Snake bites are a typical occurrence, especially in rural areas where farming and encounters between humans and snakes are common. Numerous snake species, including the dangerous Russell’s viper and the spectacled cobra, are found in Sri Lanka specifically. Depending on the type of snake bitten and the amount of venom administered, different clinical symptoms might result from a snake bite. Pain, bruising, swelling, and systemic symptoms like nausea, vomiting, and paralysis are typical ones. Treatment that is given too slowly or not at all can result in fatal consequences. Antivenom delivery, wound care, and supportive measures are all essential components of managing snake bite cases effectively. However, obstacles including a lack of access to medical facilities and less availability of antivenom sometimes make it difficult to get treatment quickly, particularly in isolated places and concern about indigenous treatment as well. Public awareness campaigns, better healthcare facilities, and epidemiology research are only a few of the measures taken against snakebite in Sri Lanka. To lessen the number of snakebites in, government institutions, medical professionals, and international organizations must work together. Better antivenom medicines and improved preventive and treatment plans for snakebite in this area require more study and funding.International Journal of Human and Health Sciences Vol. 08 No. 02 Apr’24 Page: 116-125

Variability in antivenom neutralization of Mexican viperid snake venoms.

Each year, 3,800 cases of snakebite envenomation are reported in Mexico, resulting in 35 fatalities. The only scientifically validated treatment for snakebites in Mexico is the use of antivenoms. Currently, two antivenoms are available in the market, with one in the developmental phase. These antivenoms, produced in horses, consist of F(ab')2 fragments generated using venoms from various species as immunogens. While previous studies primarily focused on neutralizing the venom of the Crotalus species, our study aims to assess the neutralization capacity of different antivenom batches against pit vipers from various genera in Mexico. We conducted various biological and biochemical tests to characterize the venoms. Additionally, we performed neutralization tests using all three antivenoms to evaluate their effectiveness against lethal activity and their ability to neutralize proteolytic and fibrinogenolytic activities. Our results reveal significant differences in protein content and neutralizing capacity among different antivenoms and even between different batches of the same product. Notably, the venom of Crotalus atrox is poorly neutralized by all evaluated batches despite being the primary cause of envenomation in the country's northern region. Furthermore, even at the highest tested concentrations, no antivenom could neutralize the lethality of Metlapilcoatlus nummifer and Porthidium yucatanicum venoms. These findings highlight crucial areas for improving existing antivenoms and developing new products. Our research reveals variations in protein content and neutralizing potency among antivenoms, emphasizing the need for consistency in venom characteristics as immunogens. While Birmex neutralizes more LD50 per vial, Antivipmyn excels in specific neutralization. The inability of antivenoms to neutralize certain venoms, especially M. nummifer and P. yucatanicum, highlights crucial improvement opportunities, given the medical significance of these species.

Role of neutrophil–lymphocyte ratio in the management of children with snake bite envenomation: A single-center prospective observational study

Background: Neutrophil–lymphocyte ratio (NLR) has recently gained popularity as a simple, inexpensive, and highly accurate inflammatory marker. When the differential count of leukocytes is checked following it is characterized by leukocytosis with an increase in neutrophil count and a decline in lymphocyte counts. This study was conducted to assess whether alteration in the differential count of leukocytes following envenomation has the ability to diagnose and predict the severity and prognosis in children and to derive a cutoff value for the same. Subjects and Methods: This prospective observational study was done in the pediatric intensive care unit (ICU) of a tertiary care hospital from July 2019 to June 2020. From the complete blood count of patients done at admission, NLR1 was calculated. All patients were treated as per the national snake bite treatment protocol. The amount of antisnake venom (ASV) given, duration of hospital stay, and ICU stay were correlated with NLR1 values. Results: An NLR value of 3.35 can predict envenomation with 95% sensitivity and 100% specificity. Similarly, an NLR value of 6.72 can predict the need for more than 10 ASV vials with a sensitivity of 93% and specificity of 97%, and an NLR value of 6.72 can predict the occurrence of major complications with a sensitivity of 89.3% and specificity of 94%. Conclusions: NLR can differentiate poisonous snake bites from nonpoisonous bites and can also predict the severity of the envenomation.

Knowledge and application of first aid and treatment of snakebites among Sri Lankan North Central Province farmers

The application of first aid for snakebites are important in rural Sri Lanka. This study aimed to assess the current level of knowledge and application of snakebite first aid. Results revealed a substantial gap between knowledge and practices. This study highlights the need for education programs to improve snakebite management

Phyto Pharmaceutical Advances on Black Turmeric as a Functional Herb

Abstract: Herbal medicine is one of humanity's oldest professions, and herbal medicine's therapeutic efficacy has been recognised since the dawn of time. In many parts of India, herbal treatments have been found to be effective in the treatment of malaria, diarrhoea, jaundice, diabetes, cough, fever, snakebite, miscarriages, and gonorrhoea. Curcuma caesia is a perennial herb with bluish-black rhizomes that is native to Northeast and Central India. In antioxidant, antiinflammatory, and human tumour cell proliferation, inhibitory effects of the rhizomes of indigenous Curcuma caesia were investigated in this work. According to Research, Inc., the global curcumin market was worth USD 46.6 million, with North America being the largest regional market and India being one of the largest curcumin producers. With a revenue-based compound yearly growth rate (CAGR) of 14.8% throughout the projection period, Europe is predicted to be the fastest-growing region, with the global market. Because of expanding scientific talent and a large network of biotechnology and food chemistry applications, curcumin quality and quantity are projected to improve in the future. The study emphasizes the importance of using the bioactive components of curcuma caesia in nanotechnology, liposomes, niosomes, micelles, and cyclodextrin.

Optimizing drug discovery for snakebite envenoming via a high-throughput phospholipase A2 screening platform.

Snakebite envenoming is a neglected tropical disease that causes as many as 1.8 million envenomings and 140,000 deaths annually. To address treatment limitations that exist with current antivenoms, the search for small molecule drug-based inhibitors that can be administered as early interventions has recently gained traction. Snake venoms are complex mixtures of proteins, peptides and small molecules and their composition varies substantially between and within snake species. The phospholipases A2 (PLA2) are one of the main pathogenic toxin classes found in medically important viper and elapid snake venoms, yet varespladib, a drug originally developed for the treatment of acute coronary syndrome, remains the only PLA2 inhibitor shown to effectively neutralise venom toxicity in vitro and in vivo, resulting in an extremely limited drug portfolio. Here, we describe a high-throughput drug screen to identify novel PLA2 inhibitors for repurposing as snakebite treatments. We present method optimisation of a 384-well plate, colorimetric, high-throughput screening assay that allowed for a throughput of ∼2,800 drugs per day, and report on the screening of a ∼3,500 post-phase I repurposed drug library against the venom of the Russell's viper, Daboia russelii. We further explore the broad-spectrum inhibitory potential and efficacy of the resulting top hits against a range of medically important snake venoms and demonstrate the utility of our method in determining drug EC50s. Collectively, our findings support the future application of this method to fully explore the chemical space to discover novel PLA2-inhibiting drugs of value for preventing severe pathology caused by snakebite envenoming.

Challenges associated with the availability, accessibility, and use of antivenoms for treating snakebite envenoming in Ghana: A MaxDiff experiment design

Successful snakebite envenoming (SBE) treatment requires safe, effective, and quality-assured antivenom products specifically tailored to combat endemic venomous snake species. This study aims to identify the challenges associated with the availability, accessibility, and use of antivenoms for treating SBE. The data for this study were obtained from a cross-sectional study involving healthcare workers from two districts (namely Afram Plains North and Afram Plains South) in the Eastern Region of Ghana. Through the MaxDiff design methodology, we quantify the challenges associated with the availability, accessibility, and use of antivenoms. Responses from a simple random sample of 203 healthcare workers were included in this study. Participants identified the high cost of antivenoms as the most challenging factor that limits the availability, accessibility, and use of antivenoms for treating SBE. Other important challenges were the lack of access to effective antivenoms in remote areas when needed and the increased use of unorthodox and harmful practices, followed by resort to unorthodox and harmful practices and the lack of effective antivenoms to address envenoming from local species in some instances. However, poor outcomes from using substandard antivenoms, stock-outs, inadequate number of manufacturers, and the resort to substandard, cheap, and harmful antivenoms were traded off. Also, poor utilization of antivenoms, suboptimal utilization of antivenoms (low quality, under-dose), use of ineffective, substandard antivenoms, and flooding of the market with products that have not been evaluated thoroughly were underscored. Our findings provide essential data to guide discussions on barriers to the availability, accessibility, and use of antivenoms for treating SBE to improve the supply of antivenoms, enhance the effectiveness of snakebite treatment, and improve patient care quality in Ghana. Multi-component strategies are needed to address the challenges identified, such as intensified advocacy, ongoing education and community engagement, healthcare worker training, and leveraging institutional and governance structures.

Exploring the Antivenom Potentials of Crinum Jagus Bulbs in the Treatment of Snake Bite from Girei, Adamawa State, Nigeria

Crinum jagus is a plant barely used in the treatment of snake bite in Nigeria and other African countries. This study focuses on evaluating the efficacy of the bulb of the plant against venom from Carpet viper snake. The plant samples were obtained, dried, blended while successive extraction was carried out using methanol. The crude methanolic extract and two distinct fractions obtained from column chromatography were used. Crystallized venom of Carpet viper was administered at varying concentrations. Forty mice were allocated into ten equal groups of four each. Mice in groups 4, 2 and 1 were given 150, 300 and 450 mg/kg of fraction 1 of Crinum jagus bulb respectively. Group 6, 5 and 7 were given same dose of fraction 2 obtained from Crinum jagus bulb. Group 7, 8 and 9 were given the crude methanolic extract at 150, 300 and 450 mg/kg, while group 10 served as the control. Apart from the Carpet viper venom that was administered intraperitonially, all others were administered orally. The Mice were observed over 24 hours before readings were taken. All the animals in the control group succumbed to envenomation within 7 to 15 minutes. In contrast, mice treated with both fractions and the crude methanolic extract exhibited significant survival rates, with survival rates increasing with higher dosages. Specifically, two mice from group 5 (F2 = 300 mg/kg), one from group 4 (F1 = 150 mg/kg), and one from group 3 (Cr = 300 mg/kg) survived the venom. The average weights of the mice across the groups varied, but no significant weight loss or adverse reactions were observed in the treated groups. These findings suggest that Crinum jagus bulb extracts possess potential antivenom properties against Carpet viper venom.

Consequences of geographical accessibility to post-exposure treatment for rabies and snakebite in Africa: a mini review.

Rabies and snakebite envenoming are two zoonotic neglected tropical diseases (NTDs) transmitted to humans by animal bites, causing each year around 179,000 deaths and are most prevalent in Asia and Africa. Improving geographical accessibility to treatment is crucial in reducing the time from bite to treatment. This mini review aims to identify and synthesize recent studies on the consequences of distance and travel time on the victims of these diseases in African countries, in order to discuss potential joint approaches for health system strengthening targeting both diseases. A literature review was conducted separately for each disease using Pubmed, Google Scholar, and snowball searching. Eligible studies, published between 2017 and 2022, had to discuss any aspect linked to geographical accessibility to treatments for either disease in Africa. Twenty-two articles (8 on snakebite and 14 on rabies) were eligible for data extraction. No study targeted both diseases. Identified consequences of low accessibility to treatment were classified into 6 categories: (1) Delay to treatment; (2) Outcome; (3) Financial impacts; (4) Under-reporting; (5) Compliance to treatment, and (6) Visits to traditional healers. Geographical access to treatment significantly influences the burden of rabies and snakebite in Africa. In line with WHO's call for integrating approaches among NTDs, there are opportunities to model disease hotspots, assess population coverage, and optimize geographic access to care for both diseases, possibly jointly. This could enhance the management of these NTDs and contribute to achieving the global snakebite and rabies roadmaps by 2030.

Phytochemical characterization and phospholipase A2 inhibitory effect of Vitex negundo L. root extracts

Ethnopharmacological relevanceSnake bites are a critical health issue in many parts of the world particularly in Asian countries lacking efficient health facilities in rural areas. Cobra is the most common snake type in Asia and is responsible for a large number of mortalities particularly in rural areas. Plants are usually considered the most effective and easy-to-approach treatment for snake bites in rural areas of various countries. Vitex negundo L. is an important medicinal plant traditionally used to treat snake bite envenomation in many countries of Asia. Aim of the studyFrom literature survey of plants traditionally used in the treatment of snake bites in Asian countries including India, Pakistan and Sri Lanka, roots of V. negundo were selected for the present study. Anti-snake venom potential of its roots was assessed through various in vitro assays targeting the phospholipase A2 enzyme. Materials and methodsV. negundo roots were sequentially extracted in different organic solvents to get fractions and in methanol to get total extract. The extracts were evaluated for phospholipase A2 (PLA2) inhibitory potential through inhibition of venom-induced hemolysis, ADP-induced platelet aggregation, PLA2-induced fatty acid hydrolysis and anticoagulant effect of cobra venom. Antioxidant power was determined using DPPH and superoxide radical scavenging assays. GC-MS and HPLC analysis was performed for the total methanol extract. ResultsStrong PLA2 inhibitory effect was observed for all the extracts. The ethyl acetate, acetone and methanol fractions significantly inhibited toxic effects of cobra venom under in vitro conditions. Radical scavenging potential of these fractions was also significantly high as compared to non-polar fractions in both DPPH and superoxide scavenging assays. Phytochemical analysis indicated high phenolic and flavonoid contents in these fractions. GC-MS and HPLC analysis of total methanol extract confirmed the presence of bis(2-ethylhexyl) phthalate, phenol, o-Guaiacol, palmitic acid-methyl ester, methyl stearate, quercetin and kaempferol in the plant. ConclusionThe study concluded that the roots of V. negundo, particularly their polar extracts, have strong PLA2 inhibitory effect against cobra venom confirming their traditional use to manage snake bites. The roots of this plant can be further studied for isolation of plant-based antisera.

Systematic review and meta-analysis on the efficacy of Indian polyvalent antivenom against the Indian snakes of clinical significance.

Snakebite in India is a severe problem as it causes a mortality rate of 58,000 and a disability rate of 140,000 every year which is the highest among any other country. Antivenom is the primary therapy for snakebite, and its manufacturing techniques have essentially stayed unaltered for over a century. Indian polyvalent antivenom, a scientifically validated medicine for treating the toxic effects of snakebites, is available against the venom of the so-called Big Four snakes namely Spectacled cobra (Naja naja), Saw-scaled viper (Echis carinatus), Russell's viper (Daboia russelli) and the Common krait (Bungarus caeruleus), responsible for majority of the deaths in India. India hosts many other species of snakes, including cobras, kraits, saw-scaled vipers, sea snakes, and pit vipers, responsible for clinically severe envenomation. Neutralization strategy has been applied to access the efficacy of antivenoms, crucial for reducing snake bite deaths and disabilities. This review aims to conduct a systematic review and meta-analysis on the neutralization efficiency of the Polyvalent Antivenom (PAV) and focus on the factors that may contribute to the poor recognition of the antivenom towards the venom toxins. Reports focusing on the investigation of antivenom efficacy were searched and collected from several databases. Preclinical studies that reported the neutralization efficacy of the commercial antivenom against the medically important snakes of India were included. The articles were screened based on the inclusion criteria and 8 studies were shortlisted for meta-analysis. Pooled proportion was calculated for the antivenom efficacy reported by the studies and was found to be statistically significant with a 95% confidence interval. The heterogenicity in the venom toxicity and neutralization potency of the antivenom was evident in the overall estimate (proportion) and individual data. We provide comprehensive evidence on antivenom efficacy against medically important snakes from various parts of India which may aid in identifying the gaps in snake envenomation therapy and the need for novel potentially improved treatment of snakebites.

Homeopathic Treatment of snakebite in dog – case report

Snakebite accidents are very common nearby forest areas. The majority of these incidents are caused by snakes of the genus Bothrops, which comprises over 45 species distributed in Central and South America. These snakes usually hunt small mammals and lizards at dusk. Snakebites from this genus generally occur in the head, limbs, and neck regions. Bothrops snakes are considered aggressive animals, characterized by inverted "V" markings along their bodies, triangular heads, smooth tails, and length ranging from 0.5m to 2m. They exhibit color variations of brown, green, and gray. Bothrops venom has necrotic, coagulating, vasculotoxic, and nephrotoxic effects, which can cause extensive necrosis at the bite site, tissue loss, and limb dysfunction, sometimes resulting in amputation. The bitten animal may experience prostration, tachypnea, localized edema and pain, petechiae, hemorrhages, and extensive necrotic areas. Hematuria, hypotension, pulmonary edema, and even death, as well as various laboratory abnormalities, may also occur. The case reported herein was attended at a veterinary clinic in Campos Altos, Minas Gerais, Brazil. It involved a mixed-breed female dog, 3 years old, weighing 4.5 kg, which had been bitten by a Bothrops snake approximately 90 minutes before. The animal presented edema and pain at the site of the bite (pelvic limb), as well as tachycardia. At once, it was administered intravenous polyvalent antiophidic serum, dexamethasone, serum, furosemide, tramadol, and ceftriaxone (antibiotic). Local treatment included corticosteroid and antibiotic ointment dressings. The animal remained stable, but after 24 hours, an extensive area with edema, hematoma, bleeding, and early necrosis at the bite site was observed, worsening with each passing hour. After 48 hours, therapy with Bothrops CH6 (5 globules every 8 hours) was initiated to reduce local effects (edema, hemorrhage, and necrosis). The animal showed rapid improvement in the appearance of the lesion, and after 5 days of treatment, there was no longer edema or bleeding, only a circumscribed cutaneous injury, which was managed with local dressings (cleaning and healing ointment). The animal recovered well without any sequelae.

Current Status and Influencing Factors of Snakebite Diagnosis and Treatment Knowledge Among Medical Staff in China: A Cross-Sectional Study.

Objectives: This study aimed to determine the current status of the knowledge of diagnosis and treatment of snakebites among medical staff in China and its influencing factors. Methods: A cross-sectional survey of 12,581 medical staff was conducted in 12 provinces in China between June 2022 and February 2023. We analyzed the results using descriptive statistics, T-tests or analysis of variance, and a generalized linear model. Results: The average score of snakebite diagnosis and treatment knowledge among medical staff in China was 3.15 ± 2.15 out of a total score of 12. Through a generalized linear regression model, we found that gender, occupation, region, hospital level, work department, work tenure, training received in the diagnosis and treatment of snakebite, experience in snakebite diagnosis and treatment, availability of antivenom in the unit, and self-evaluation of snakebite treatment ability all affected the medical staff's scores of snakebite diagnosis and treatment knowledge. Conclusion: The knowledge level of snakebite diagnosis and treatment among Chinese medical staff is generally low, so it is imperative to conduct standardized snakebite diagnosis and treatment training for medical staff.

ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming.

Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom-polyclonal antibodies isolated from the plasma of hyperimmunised animals-which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability.

Acetylcholine-Binding Protein Affinity Profiling of Neurotoxins in Snake Venoms with Parallel Toxin Identification

Snakebite is considered a concerning issue and a neglected tropical disease. Three-finger toxins (3FTxs) in snake venoms primarily cause neurotoxic effects since they have high affinity for nicotinic acetylcholine receptors (nAChRs). Their small molecular size makes 3FTxs weakly immunogenic and therefore not appropriately targeted by current antivenoms. This study aims at presenting and applying an analytical method for investigating the therapeutic potential of the acetylcholine-binding protein (AChBP), an efficient nAChR mimic that can capture 3FTxs, for alternative treatment of elapid snakebites. In this analytical methodology, snake venom toxins were separated and characterised using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and high-throughput venomics. By subsequent nanofractionation analytics, binding profiling of toxins to the AChBP was achieved with a post-column plate reader-based fluorescence-enhancement ligand displacement bioassay. The integrated method was established and applied to profiling venoms of six elapid snakes (Naja mossambica, Ophiophagus hannah, Dendroaspis polylepis, Naja kaouthia, Naja haje and Bungarus multicinctus). The methodology demonstrated that the AChBP is able to effectively bind long-chain 3FTxs with relatively high affinity, but has low or no binding affinity towards short-chain 3FTxs, and as such provides an efficient analytical platform to investigate binding affinity of 3FTxs to the AChBP and mutants thereof and to rapidly identify bound toxins.

Differentiating Dyes: A Spectroscopic Investigation into the Composition of Scarlet Bloodroot (Haemodorum coccineum R.Br.) Rhizome.

Haemodorum coccineum, commonly known as scarlet bloodroot, is a plant native to New Guinea and the northern most parts of Australia. The highly coloured H. coccineum is used by communities in Larrakia country for dyeing garments and occasionally to treat snake bites. Previous studies into H. coccineum have focused on its taxonomic classification, with this being the first evaluation of the chemical composition of the plant. Haemodoraceae plants are reported to contain phenylphenalenones (PhPs), which are highly conjugated polycyclic oxygenated aromatic hydrocarbons. We report the characterisation of 20 compounds extracted from the rhizome of H. coccineum: four sugars and 16 compounds belonging to the PhP family. The compounds include five aglycones and seven glycosylated compounds, of which four contain malonate esters in their structures. Characterisation of these compounds was achieved through 1D and 2D NMR, MS analysis and comparison to the known phytochemistry of other species from the Haemodorum genus. Preliminary anti-microbial activity of the crude extract shows significant inhibition of the growth of both gram-positive and gram-negative bacteria, but no activity against Candida albicans.

Identification of antioxidant compounds from the stem bark fraction of Strychnosinnocua Del.

Strychnos innocua is a shrub that is native to the tropic region of the world. It is used locally as food, medicine, fuel, and as an insect repellent. Extracts of this plant are used as an aphrodisiac and in the treatment of gonorrhoea, snakebite and dysentery. It has been proven to possess antioxidant and antimicrobial activities. There is little or no information on the identification and pharmacological activities of the chemical composition of the stem bark of this plant. The aim of this work is to identify the chemical compounds present in this plant and determine their antioxidant activities. The ethyl acetate extract of this plant was subjected to purification techniques which lead to the isolation of fraction SI1. The chemical composition of the fraction SI1 was then determined using gas chromatography–mass spectrometry (GCMS). Fractions SI1 was then analysed for its free radical scavenging potential using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and Ferric ion reducing antioxidant property (FRAP) assay. A total of thirty compounds were identified with the major compounds being Cyclohex-2-enone,3-)2H-tetrazol-5-yl amino) (15.83 %), N-hexadecanoic acid (38.21%), N-hexadecanoic acid (38.21%), Octadecanoic acid (13.41%), Cis-vaccenic acid (13.41 %). The DPPH radical scavenging potential of the fraction SI1 compares well with the standard, ascorbic acid with IC50 of 3.71 ± 0.31 μg/mL and 1.68 ± 1.27 μg/mL, respectively. At 500 μg/mL, the antioxidant potential of the standard ascorbic acid was 96.9% whereas the fraction SI1 had 62.1% free DPPH radical scavenging activity. The half-maximal inhibitory concentration (IC50) of SI1 observed is 0.0488 ± 0.01 nm, and 0.5375 ± 0.01 nm and for ascorbic acid. In conclusion, fraction SI1 can act as an antioxidant supplement for various applications in diverse fields of food, cosmetics, and medicine. Several compounds contained in the fraction SI1 may have contributed to the antioxidant properties of the fraction. The compounds identified could be useful precursors for the synthesis of therapeutic drugs associated with the treatment of human and animal diseases.

Analysis of the heavy metal contents' effect on steroidal saponins and the anti-breast cancer activity of Paris polyphylla var. yunnanensis.

Background: P. polyphylla var. yunnanensis, as a near-threatened and ethnic medicine in China, used to be a key ingredient in traditional Chinese medicine in treatment of traumatic injuries, sore throat, snakebites, and convulsions for thousands of years. However, there were no reports on the inverse relationship between the contents of heavy metals and saponins and its anti-breast cancer pharmacological activity in P. polyphylla var. yunnanensis. Methods: The present study aimed to reveal the characteristics of heavy metal contents and saponins and its anti-breast cancer pharmacological activity and their interrelationships in P. polyphylla var. yunnanensis from different production areas. The contents of heavy metal and steroidal saponins in P. polyphylla var. yunnanensis were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) and the high-performance liquid chromatography technique, respectively. The Pearson correlation was used to study the correlation between saponins and heavy metals. 4T1 mouse mammary tumor cells were selected and cultivated for antitumor studies in vitro. Cell Counting Kit-8 (CCK-8) assay, Hoechst staining, and flow cytometry analysis were used for the examination of the proliferation and apoptosis of 4T1 tumor cells. Mouse breast cancer 4T1 cells were subcutaneously injected into BALB/c mice to construct a tumor model to explore the in vivo inhibitory effect on breast cancer. TUNEL assay and immunohistochemistry were used for the examination of the effect of P. polyphylla var. yunnanensis from different origins on cancer cell proliferation and apoptosis induction in 4T1 tumor mice. Results: Heavy metal contents were highly correlated with the content of steroidal saponins. The overall content of 10 metals in the three producing origins was of the order C3 >C2 >C1. The total content of eight steroidal saponins in the extracts of P. polyphylla var. yunnanensis from three different origins was C1 >C2 >C3. The Pearson correlation study showed that in all of the heavy metals, the contents of Cd and Ba were positively correlated with the main steroidal saponins in P. polyphylla var. yunnanensis, while Al, Cr, Cu, Fe, Zn, As, Hg, and Pb showed a negative correlation. In vitro experiments showed that the extracts of P. polyphylla var. yunnanensis from three origins could inhibit the proliferation and induce cell apoptosis of 4T1 cells in a concentration- and time-dependent manner, especially in the C1 origin. In vivo experiments showed that the extract of P. polyphylla var. yunnanensis from the three origins could inhibit the growth of tumors and induce the apoptosis of tumor cells. In the three origins, C1 origin had the lowest total heavy metal level but the highest total steroidal saponin level. Therefore, it showed a better effect in reducing the expression of the human epidermal growth factor receptor 2 (HER2) and Kiel 67 (Ki67) and increasing the expression of p53 in tumor tissues compared to the other origins. In conclusion, in the three origins, C1 origin exhibits antitumor pharmacological effects in vivo and in vitro which are better than those in the other origins. Conclusion: In this study, we found that with the increase of the heavy metal content, the content of steroid saponins and anti-breast cancer activity decreased. The results showed that the high content of the total heavy metals may not be conducive to the accumulation of steroidal saponins in P. polyphylla var. yunnanensis and lead to the low anti-breast cancer activity. The results of this study suggest that the content of heavy metals should be controlled in the artificial cultivation process of P. polyphylla var. yunnanensis.